Hyaluronan-CD44 interaction with leukemia-associated RhoGEF and epidermal growth factor receptor promotes Rho/Ras co-activation, phospholipase C epsilon-Ca2+ signaling, and cytoskeleton modification in head and neck squamous cell carcinoma cells

In this study we have examined the interaction of CD44 (a major hyaluronan (HA) receptor) with a RhoA-specific guanine nucleotide exchange factor (leukemia-associated RhoGEF (LARG)) in human head and neck squamous carcinoma cells (HNSCC-HSC-3 cell line). Immunoprecipitation and immunoblot analyses indicate that CD44 and the LARG protein are expressed in HSC-3 cells and that these two proteins are physically associated as a complex. HA-CD44 binding induces LARG-specific RhoA signaling and phospholipase C epsilon (PLC epsilon) activity. In particular, the activation of RhoA-PLC epsilon by HA stimulates inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, and the up-regulation of Ca2+/calmodulin-dependent kinase II (CaMKII), leading to phosphorylation of the cytoskeletal protein, filamin. The phosphorylation of filamin reduces its interaction with filamentous actin, promoting tumor cell migration. The CD44-LARG complex also interacts with the EGF receptor (EGFR). Most importantly, the binding of HA to the CD44-LARG-EGFR complex activates the EGFR receptor kinase, which in turn promotes Ras-mediated stimulation of a downstream kinase cascade including the Raf-1 and ERK pathways leading to HNSCC cell growth. Using a recombinant fragment of LARG (the LARG-PDZ domain) and a binding assay, we have determined that the LARG-PDZ domain serves as a direct linker between CD44 and EGFR. Transfection of the HSC-3 cells with LARG-PDZcDNA significantly reduces LARG association with CD44 and EGFR. Overexpression of the LARG-PDZ domain also functions as a dominant-negative mutant (similar to the PLC/Ca2+-calmodulin-dependent kinase II (CaMKII) and EGFR/MAPK inhibitor effects) to block HA/CD44-mediated signaling events (e.g. EGFR kinase activation, Ras/RhoA co-activation, Raf-ERK signaling, PLC epsilon-mediated inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, CaMKII activity, filamin phosphorylation, and filamin-actin binding) and to abrogate tumor cell growth/migration. Taken together, our findings suggest that CD44 interaction with LARG and EGFR plays a pivotal role in Rho/Ras co-activation, PLC epsilon-Ca2+ signaling, and Raf/ERK up-regulation required for CaMKII-mediated cytoskeleton function and in head and neck squamous cell carcinoma progression.     

The cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) contributes to epidermal growth factor receptor-mediated cell migration

Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.     

EGF AND TGF-alpha motogenic activities are mediated by the EGF receptor via distinct matrix-dependent mechanisms

EGF and TGF-alpha induce an equipotent stimulation of fibroblast migration and proliferation. In spite of their homologous structure and ligation by the same receptor (EGFR), we report that their respective motogenic activities are mediated by different signal transduction intermediates, with p70(S6K) participating in EGF signalling and phospholipase Cgamma in TGF-alpha signalling. We additionally demonstrate that EGF and TGF-alpha motogenic activities may be resolved into two stages: (a) cell "activation" by a transient exposure to either cytokine, and (b) the subsequent "manifestation" of an enhanced migratory phenotype in the absence of cytokine. The cell activation and manifestation stages for each cytokine are mediated by distinct matrix-dependent mechanisms: motogenetic activation by EGF requires the concomitant functionality of EGFR and the hyaluronan receptor CD44, whereas activation by TGF-alpha requires EGFR and integrin alphavbeta3. Manifestation of elevated migration no longer requires the continued presence of exogenous cytokine and functional EGFR but does require the above mentioned matrix receptors, as well as their respective ligands, i.e., hyaluronan in the case of EGF, and vitronectin in the case of TGF-alpha. In contrast, the mitogenic activities of EGF and TGF-alpha are independent of CD44 and alphavbeta3 functionality. These results demonstrate clear qualitative differences between EGF and TGF-alpha pathways and highlight the importance of the extracellular matrix in regulating cytokine bioactivity.     

The interaction between CD44 on tumour cells and hyaluronan under physiologic flow conditions: implications for metastasis formation

The adhesion of tumour cells to the endothelial cells of blood vessels of the microcirculation represents a crucial step in haematogenous metastasis formation. Similar to leukocyte extravasation, selectins mediate initial tumour cell rolling on endothelium. An additional mechanism of leukocyte adhesion to endothelial cells is mediated by hyaluronan (HA). However, data on the interaction of tumour cells with hyaluronan under shear stress are lacking. The expression of the hyaluronan binding protein CD44 on tumour cell surfaces was evaluated using flow cytometry. The adhesion of tumour cells to HA with regard to adhesive events and rolling velocity was determined in flow assays in the human small cell lung cancer (SCLC) cell lines SW2, H69, H82, OH1 and OH3, the colon carcinoma cell line HT29 and the melanoma cell line MeWo. Hyaluronan deposition in human and mouse lung blood vessels was histochemically determined. MeWo adhered best to HA followed by HT29. SCLC cell lines showed the lowest CD44 expression on the cell surface and lowest number of adhesive events. While hyaluronan was deposited in patches in the microvasculature of the alveolar septum in the human lung, it was only present in the periarterial space in the mouse lung. Certain tumour entities bind to HA under physiological shear stresses so that HA can be considered a further ligand for cell extravasation in haematogenous metastasis. As hyaluronan is deposited within the pulmonary microvasculature, it may well serve as a ligand for its binding partner CD44, which is expressed by many tumour cells.     

Growth stimulation of non-small cell lung cancer cell lines by antibody against epidermal growth factor receptor promoting formation of ErbB2/ErbB3 heterodimers

Antibodies are the most rapidly expanding class of human therapeutics, including their use in cancer therapy. Monoclonal antibodies (mAb) against epidermal growth factor (EGF) receptor (EGFR) generated for cancer therapy block the binding of ligand to various EGFR-expressing human cancer cell lines and abolish ligand-dependent cell proliferation. In this study, we show that our mAb against EGFRs, designated as B4G7, exhibited a growth-stimulatory effect on various human cancer cell lines including PC-14, a non-small cell lung cancer cell line; although EGF exerted no growth-stimulatory activity toward these cell lines. Tyrosine phosphorylation of EGFRs occurred after treatment of PC-14 cells with B4G7 mAb, and it was completely inhibited by AG1478, a specific inhibitor of EGFR tyrosine kinase. However, this inhibitor did not affect the B4G7-stimulated cell growth, indicating that the growth stimulation by B4G7 mAb seems to be independent of the activation of EGFR tyrosine kinase. Immunoprecipitation with anti-ErbB3 antibody revealed that B4G7, but not EGF, stimulated heterodimerization between ErbB2 and ErbB3. ErbB3 was tyrosine phosphorylated in the presence of B4G7 but not in the presence of EGF. Further, the phosphorylation and B4G7-induced increase in cell growth were inhibited by AG825, a specific inhibitor of ErbB2. These results show that the ErbB2/ErbB3 dimer functions to promote cell growth in B4G7-treated cells. Changes in receptor-receptor interactions between ErbB family members after inhibition of one of its members are of potential importance in optimizing current EGFR family-directed therapies for cancer.     

Hyaluronan constitutively regulates ErbB2 phosphorylation and signaling complex formation in carcinoma cells

Hyaluronan is enriched in many types of human cancers, and manipulations of hyaluronan expression or interactions have a major influence on tumor progression in animal models. Increased ErbB2 activity is characteristic of several cancers and is responsible for many aspects of malignant cell behavior in these cancers. In this study we show that constitutively high levels of active, i.e. autophosphorylated, ErbB2 in HCT116 colon carcinoma cells and TA3/St mammary carcinoma cells are dependent on endogenous hyaluronan-CD44 interaction. Dependence on hyaluronan-CD44 interaction was demonstrated by the administration of hyaluronan oligomers, experimentally induced expression of soluble CD44, and small interfering RNA knockdown of CD44 expression. On the other hand, increasing hyaluronan production by overexpression of hyaluronan synthase 2 or emmprin causes elevated ErbB2 phosphorylation in MCF-7 mammary carcinoma cells, which normally exhibit low levels of ErbB2 activity. Furthermore, in HCT116 and TA3/St cells, inhibition of endogenous hyaluronan-CD44 interaction causes disassembly of a constitutive, lipid raft-associated, signaling complex containing phosphorylated ErbB2, CD44, ezrin, phosphoinositide 3-kinase, and the chaperone molecules, Hsp90 and cdc37. Stimulation of hyaluronan production in MCF-7 cells induces assembly of this complex. We conclude that hyaluronan regulates ErbB2 activity and its interactions with other signaling factors in carcinoma cells.     

Regulation of the EGFR/ErbB signalling by clathrin in response to various ligands in hepatocellular carcinoma cell lines

Membrane receptor intracellular trafficking and signalling are frequently altered in cancers. Our aim was to investigate whether clathrin‐dependent trafficking modulates signalling of the ErbB receptor family in response to amphiregulin (AR), EGF, heparin‐binding EGF‐like growth factor (HB‐EGF) and heregulin‐1β (HRG). Experiments were performed using three hepatocellular carcinoma (HCC) cell lines, Hep3B, HepG2 and PLC/PRF/5, expressing various levels of EGFR, ErbB2 and ErbB3. Inhibition of clathrin‐mediated endocytosis (CME), by down‐regulating clathrin heavy chain expression, resulted in a cell‐ and ligand‐specific pattern of phosphorylation of the ErbB receptors and their downstream effectors. Clathrin down‐regulation significantly decreased the ratio between phosphorylated EGFR (pEGFR) and total EGFR in all cell lines when stimulated with AR, EGF, HB‐EGF or HRG, except in HRG‐stimulated Hep3B cells in which pEGFR was not detectable. The ratio between phosphorylated ErbB2 and total ErbB2 was significantly decreased in clathrin down‐regulated Hep3B cells stimulated with any of the ligands, and in HRG‐stimulated PLC/PRF/5 cells. The ratio between phosphorylated ErbB3 and total ErbB3 significantly decreased in clathrin down‐regulated cell lines upon stimulation with EGF or HB‐EGF. STAT3 phosphorylation levels significantly increased in all cell lines irrespective of stimulation, while that of AKT remained unchanged, except in AR‐stimulated Hep3B and HepG2 cells in which pAKT was significantly decreased. Finally, ERK phosphorylation was insensitive to clathrin inhibition. Altogether, our observations indicate that clathrin regulation of ErbB signalling in HCC is a complex process that likely depends on the expression of ErbB family members and on the autocrine/paracrine secretion of their ligands in the tumour environment.     

SMURF1 Plays a role in EGF-induced breast cancer cell migration and invasion

Epidermal growth factor (EGF) is a well-known growth factor that induces cancer cell migration and invasion. Previous studies have shown that SMAD ubiquitination regulatory factor 1 (SMURF1), an E3 ubiquitin ligase, regulates cell motility by inducing RhoA degradation. Therefore, we examined the role of SMURF1 in EGF-induced cell migration and invasion using MDA-MB-231 cells, a human breast cancer cell line. EGF increased SMURF1 expression at both the mRNA and protein levels. All ErbB family members were expressed in MDA-MB-231 cells and receptor tyrosine kinase inhibitors specific for the EGF receptor (EGFR) or ErbB2 blocked the EGF-mediated induction of SMURF1 expression. Within the signaling pathways examined, ERK1/2 and protein kinase C activity were required for EGF-induced SMURF1 expression. The overexpression of constitutively active MEK1 increased the SMURF1 to levels similar to those induced by EGF. SMURF1 induction by EGF treatment or by the overexpression of MEK1 or SMURF1 resulted in enhanced cell migration and invasion, whereas SMURF1 knockdown suppressed EGF- or MEK1-induced cell migration and invasion. EGF treatment or SMURF1 overexpression decreased the endogenous RhoA protein levels. The overexpression of constitutively active RhoA prevented EGF- or SMURF1-induced cell migration and invasion. These results suggest that EGFinduced SMURF1 plays a role in breast cancer cell migration and invasion through the downregulation of RhoA.     

E5 Protein of Human Papillomavirus Type 16 Protects Human Foreskin Keratinocytes from UV B-Irradiation-Induced Apoptosis

The human papillomavirus type 16 (HPV16) E5 protein associates with the epidermal growth factor receptor (EGFR) and enhances the activation of the EGFR after stimulation by EGF in human keratinocytes. Phosphatidylinositol 3-kinase (PI3K) and ERK1/2 mitogen-activated protein kinase (ERK1/2 MAPK), two signal molecules downstream of the EGFR, have been recognized as participants in two survival signal pathways in response to stress. The fact that E5 can enhance EGFR activation suggests that E5 might act as a survival factor. To test this hypothesis, the apoptotic response of UV B-irradiated primary keratinocytes infected with either control retrovirus, LXSN, or HPV16 2E5-expressing recombinant retrovirus was quantitated. Under the same conditions, LXSN-infected cells showed extensive apoptosis, while E5-expressing cells demonstrated a significant reduction in UV B-irradiation-induced apoptosis. The E5-mediated protection against apoptosis was blocked by wortmannin and PD98059, specific inhibitors of the PI3K and ERK1/2 MAPK pathways, respectively, suggesting that the PI3K and ERK1/2 MAPK pathways are involved in this process. Western blot analysis showed that Akt (also named protein kinase B), which is a downstream effector of PI3K, and ERK1/2 MAPK were activated by EGF. When cells were stimulated by EGF and irradiated by UV B, the levels of phospho-Akt and phospho-ERK1/2 activated by EGF in E5-expressing cells were about twofold greater than those in LXSN-infected cells. Two other UV-activated stress pathways, p38 and JNK, were activated to the same level during UV B irradiation in both LXSN-infected cells and E5-expressing cells, indicating that E5 protein did not affect these two pathways. After UV B irradiation, p53 was activated in both LXSN-infected cells and E5-expressing cells, and cell cycle analysis showed that nearly all cells in both cell populations were growth arrested. These data suggest that unlike HPV16 E6, which blocks apoptosis by inactivation of p53, HPV16 E5 protects cells from apoptosis by enhancing the PI3K-Akt and ERK1/2 MAPK signal pathways.     

Signal transduction of betacellulin in growth and migration of vascular smooth muscle cells

Epidermal growth factor (EGF) family ligands have been implicated in cardiovascular diseases because of their enhanced expression in vascular lesions and their promoting effects on growth and migration of vascular smooth muscle cells (VSMCs). Betacellulin (BTC), a novel EGF family ligand, has been shown to be expressed in atherosclerotic lesions and to be a potent growth factor of VSMCs. However, the molecular mechanisms downstream of BTC involved in mediating vascular remodeling remain largely unknown. Therefore, the aim of this study was to examine the effects of BTC on signal transduction, growth, and migration in VSMCs. We found that BTC stimulated phosphorylation of EGF receptor (EGFR) at Tyr1068, which was completely blocked by an EGFR kinase inhibitor, AG-1478. BTC also phosphorylated ErbB2 at Tyr877, Tyr1112, and Tyr1248 and induced association of ErbB2 with EGFR, suggesting their heterodimerization in VSMCs. In postreceptor signal transduction, BTC stimulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2, Akt, and p38 mitogen-activated protein kinase (MAPK). Moreover, BTC stimulated proliferation and migration of VSMCs. ERK and Akt inhibitors suppressed migration markedly and proliferation partially, whereas the p38 inhibitor suppressed migration partially but not proliferation. In addition, we found the presence of endogenous BTC in conditioned medium of VSMCs and an increase of BTC on angiotensin II stimulation. In summary, BTC promotes growth and migration of VSMCs through activation of EGFR, ErbB2, and downstream serine/threonine kinases. Together with the expression and processing of endogenous BTC in VSMCs, our results suggest a critical involvement of BTC in vascular remodeling. epidermal growth factor receptors; ErbB2; migration; signal transduction     

P2Y2 Nucleotide Receptors Mediate Metalloprotease-dependent Phosphorylation of Epidermal Growth Factor Receptor and ErbB3 in Human Salivary Gland Cells

The G protein-coupled receptor P2Y₂ nucleotide receptor (P2Y₂R) has been shown to be up-regulated in a variety of tissues in response to stress or injury. Recent studies have suggested that P2Y₂Rs may play a role in immune responses, wound healing, and tissue regeneration via their ability to activate multiple signaling pathways, including activation of growth factor receptors. Here, we demonstrate that in human salivary gland (HSG) cells, activation of the P2Y₂R by its agonist induces phosphorylation of ERK1/2 via two distinct mechanisms, a rapid, protein kinase C-dependent pathway and a slower and prolonged, epidermal growth factor receptor (EGFR)-dependent pathway. The EGFR-dependent stimulation of UTP-induced ERK1/2 phosphorylation in HSG cells is inhibited by the adamalysin inhibitor tumor necrosis factor-α protease inhibitor or by small interfering RNA that selectively silences ADAM10 and ADAM17 expression, suggesting that ADAM metalloproteases are required for P2Y₂R-mediated activation of the EGFR. G protein-coupled receptors have been shown to promote proteolytic release of EGFR ligands; however, neutralizing antibodies to known ligands of the EGFR did not inhibit UTP-induced EGFR phosphorylation. Immunoprecipitation experiments indicated that UTP causes association of the EGFR with another member of the EGF receptor family, ErbB3. Furthermore, stimulation of HSG cells with UTP induced phosphorylation of ErbB3, and silencing of ErbB3 expression inhibited UTP-induced phosphorylation of both ErbB3 and EGFR. UTP-induced phosphorylation of ErbB3 and EGFR was also inhibited by silencing the expression of the ErbB3 ligand neuregulin 1 (NRG1). These results suggest that P2Y₂R activation in salivary gland cells promotes the formation of EGFR/ErbB3 heterodimers and metalloprotease-dependent neuregulin 1 release, resulting in the activation of both EGFR and ErbB3.     

ErbB2 is necessary for induction of carcinoma cell invasion by ErbB family receptor tyrosine kinases

The epidermal growth factor (EGF) family of tyrosine kinase receptors (ErbB1, -2, -3, and -4) and their ligands are involved in cell differentiation, proliferation, migration, and carcinogenesis. However, it has proven difficult to link a given ErbB receptor to a specific biological process since most cells express multiple ErbB members that heterodimerize, leading to receptor cross-activation. In this study, we utilize carcinoma cells depleted of ErbB2, but not other ErbB receptor members, to specifically examine the role of ErbB2 in carcinoma cell migration and invasion. Cells stimulated with EGF-related peptides show increased invasion of the extracellular matrix, whereas cells devoid of functional ErbB2 receptors do not. ErbB2 facilitates cell invasion through extracellular regulated kinase (ERK) activation and coupling of the adaptor proteins, p130CAS and c-CrkII, which regulate the actin-myosin cytoskeleton of migratory cells. Overexpression of ErbB2 in cells devoid of other ErbB receptor members is sufficient to promote ERK activation and CAS/Crk coupling, leading to cell migration. Thus, ErbB2 serves as a critical component that couples ErbB receptor tyrosine kinases to the migration/invasion machinery of carcinoma cells.     

β-Heregulin impairs EGF induced PLC-γ1 signalling in human breast cancer cells

The interplay of ErbB receptor homo- and heterodimers plays a crucial role in the pathology of breast cancer since activated signal transduction cascades coordinate proliferation, survival and migration of cells. EGF and β-Heregulin are well characterised ligands known to induce ErbB homo- and heterodimerisation, which have been associated with disease progression. In the present study, we investigated the impact of both factors on the migration of MDA-NEO and MDA-HER2 human breast cancer cells. MDA-NEO cells are positive for EGFR and HER3, while MDA-HER2 cells express EGFR, HER2 and HER3. Cell migration analysis revealed that β-Heregulin potently impaired EGF induced migration in both cell lines. Western blot studies showed that both ErbB receptor and PLC-γ1 tyrosine phosphorylation levels were diminished in EGF and β-Heregulin co-treated MDA-NEO and MDA-HER2 cells, which was further correlated to a significantly impaired calcium influx. Our data indicate that EGF and HRG may interfere with each other for receptor binding and dimerisation, which ultimately has an impact on signalling outcome.     

Heregulin-mediated ErbB2-ERK signaling activates hyaluronan synthases leading to CD44-dependent ovarian tumor cell growth and migration

Heregulin (HRG)-induced cell responses are mediated by the ErbB family of tyrosine kinase receptors. In this study we have investigated HRG activation of ErbB2, extracellular signal-regulated kinase (ERK) signaling, and their role in regulating hyaluronan synthase (HAS) activity in human ovarian tumor cells (SK-OV-3.ipl cells). Immunological and biochemical analyses indicate that ErbB2, ErbB3, and ErbB4 are all expressed in SK-OV-3.ipl cells and that ErbB4 (but not ErbB3) is physically linked to ErbB2 following HRG stimulation. Furthermore, our data indicate that the HRG-induced ErbB2.ErbB4 complexes stimulate ErbB2 tyrosine kinase, which induces both ERK phosphorylation and kinase activity. The activated ERK then increases the phosphorylation of HAS1, HAS2, and HAS3. Consequently, all three HAS isozymes are activated resulting in hyaluronan (HA) production. Because HRG-mediated HAS isozyme phosphorylation/activation can be effectively blocked by either AG825 (an ErbB2 inhibitor) or thiazolidinedione compound (an ERK blocker), we conclude that ErbB2-ERK signaling and HAS isozyme phosphorylation/HA production are functionally coupled in SK-OV-3.ipl cells. HRG also promotes HA- and CD44-dependent oncogenic events (e.g. CD44-Cdc42 association, p21-activated kinase 1 activation, and p21-activated kinase 1-filamin complex formation) and tumor cell-specific behaviors in an ErbB2-ERK signaling-dependent manner. Finally, we have found that the down-regulation of HAS isozyme expression (by transfecting cells with HAS1/HAS2/HAS3-specific small interfering RNAs) not only inhibits HRG-mediated HAS phosphorylation/activation and HA production but also impairs CD44-specific Cdc42-PAK1/filamin signaling, cytoskeleton activation and tumor cell behaviors. Taken together, these findings clearly indicate that HRG activation of ErbB2-ERK signaling modulates HAS phosphorylation/activation and HA production leading to CD44-mediated oncogenic events and ovarian cancer progression.     

Versican G3 domain regulates neurite growth and synaptic transmission of hippocampal neurons by activation of epidermal growth factor receptor

Versican is one of the major extracellular matrix (ECM) proteins in the brain. ECM molecules and their cleavage products critically regulate the growth and arborization of neurites, hence adjusting the formation of neural networks. Recent findings have revealed that peptide fragments containing the versican C terminus (G3 domain) are present in human brain astrocytoma. The present study demonstrated that a versican G3 domain enhanced cell attachment, neurite growth, and glutamate receptor-mediated currents in cultured embryonic hippocampal neurons. In addition, the G3 domain intensified dendritic spines, increased the clustering of both synaptophysin and the glutamate receptor subunit GluR2, and augmented excitatory synaptic activity. In contrast, a mutated G3 domain lacking the epidermal growth factor (EGF)-like repeats (G3deltaEGF) had little effect on neurite growth and glutamatergic function. Treating the neurons with the G3-conditioned medium rapidly increased the levels of phosphorylated EGF receptor (pEGFR) and phosphorylated extracellular signal-regulated kinase (pERK), indicating an activation of EGFR-mediated signaling pathways. Blockade of EGFR prevented the G3-induced ERK activation and suppressed the G3-provoked enhancement of neurite growth and glutamatergic function but failed to block the G3-mediated enhancement of cell attachment. These combined results indicate that the versican G3 domain regulates neuronal attachment, neurite outgrowth, and synaptic function of hippocampal neurons via EGFR-dependent and -independent signaling pathway(s). Our findings suggest a role for ECM proteolytic products in neural development and regeneration.     

Expression of Recombinant Hyaluronan Synthase (HAS) Isoforms in CHO Cells Reduces Cell Migration and Cell Surface CD44

In the present study we investigated the functional properties of the three recombinant hyaluronan synthases (HAS proteins) HAS1, HAS2, and HAS3. HAS3-transfected CHO clones exhibited the highest hyaluronan polymerization rate followed by HAS2 transfectants which were more catalytically active than HAS1 transfectants. In living cells all three HAS proteins synthesized hyaluronan chains of high molecular weight (larger than 3.9 x 10(6)). In vitro, the HAS2 isoform produced hyaluronan chains of a molecular weight larger than 3.9 x 10(6), whereas HAS3 produced polydisperse hyaluronan (molecular weight 0.12-1 x 10(6)), and HAS1 synthesized much shorter chains of an average molecular weight of 0.12 x 10(6). Thus, each HAS protein may interact with different cytoplasmic proteins which may influence their catalytic activity. CHO transfectants with the ability to synthesize about 1 microgram hyaluronan/1 x 10 (5) cells/24 h were surrounded by hyaluronan-containing coats, whereas transfectants generating about 4-fold lower amounts of hyaluronan formed coats only in the presence of chondroitin sulfate proteoglycan. An inverse correlation between hyaluronan production on the one hand and cell migration and cell surface CD44 expression on the other was found; a 4-fold lower migration and a 2-fold decrease of cell surface CD44 receptors was seen when hyaluronan production increased 1000-fold over the level in the untransfected cells. The inverse relationships between hyaluronan production and migration and CD44 expression of cells are of importance for the regulation of cell-extracellular matrix interactions.